The long-term goal of this research is to understand the mechanisms of the evolutionary diversification of immunoglobulin (Ig) genes. Unique methods will be used to identify Ig and Ig-like genes in species that represent critical points in the evolutionary radiation of vertebrates. Studies of the Ig genes in Heterodontus will focus on the role of somatic rearrangement/recombination and the generation of antibody diversity in both heavy and light chain gene families. Parallel studies with other lower vertebrate species, including a representative teleost and chondrostean, will determine the evolutionary distribution of the T cell receptor-like pattern of Ig gene organization observed in Heterodontus. Possible developmental stage-specific utilization o Ig genes in several different configurations will be examined. The relationship of gene organization to the Ig class shift, a different type of somatic rearrangement process, will be characterized in another elasmobranch. The next major objective will imply unique approaches to identify and characterize Ig genes in the more phylogenetically distant jawless vertebrates, represented by Petromyzon and Eptatretus. Purification of Ig-like heterodimers, production of antisera to heterodimer subunits, expression library screening, heterologous RNA/DNA hybridization, RNA:RNA-based hybridization methods and oligodeoxyribonucleotide probes complementing different peptide regions will be employed. The structure and organization of Ig genes, sites of Ig production and the relative role of somatic vs. germline mechanisms in generating antibody diversity will be addressed. Information gained in these studies will be used to develop strategies for identifying and isolating putative Ig-like heterodimers in protochordates, represented by Ciona. Screening of both cDNA and genomic expression libraries with antisera to intact molecules and heterodimeric subunits as well as different types of heterologous screening will be used. In all of these studies, data will be analyzed by sequence comparison, identification of regulatory and recombination elements and detailed examination of Ig gene organization and chromosomal linkage. Taken together, the studies will provide a fundamental understanding of the phylogenetic diversification of the rearranging multigene families of antigen receptors that are involved in the development of neoplasias, immunodeficiencies and other disorders of the immune system.